This invention is in the general field of methods, reagents, and apparatus for authenticating or monitoring sample composition.
Authenticating and monitoring products to discriminate between very similar complex mixtures is useful for various reasons. First, the use of counterfeit substances (e.g., misbranded material from a competitor or misformulated material from a licensee/franchisee) should be detected to preserve the integrity of a brand.
Characteristics of a product can be used to identify its lot. Similar methods can be used in quality control tests. Also, product counterfeiting raises serious health and safety issues. In 1995, a counterfeit-labeled version of infant formula reportedly was distributed to 15 states in the continental United States. Counterfeit wine, spirits, perfume, infant formula, soft drinks, cosmetics, and pharmaceuticals are estimated to cost United States businesses 200 billion dollars per year (xe2x80x9cThe Boston Phoenix,xe2x80x9d Section One, Dec. 2, 1994).
It is important to develop rapid, cost effective, and enforceable methods to identify fraudulent or tampered products. It is also important to determine manufacturing compliance using automated methods to decrease the amount of time spent identifying fraudulent products. It is desirable to minimize the time required from highly skilled researchers and technicians to conduct and record the results of on-line, off-line, and off-the-shelf product authenticity/compliance tests.
There have been attempts to determine product (e.g., infant formula) authenticity by protein electrophoresis, which requires substantial time (and expense) for set up and analysis. In other industries, e.g. wine and spirits, Fourier-transform infrared analysis, gas chromatography, pH, Raman spectroscopy and other analytical methods have been used or proposed for product authentication (Constant et al., Differentiation of Alcoholic Beverages FT-IR Spectra. An Original Multivariate Approach, ACS Abstract presented at 208th ACS National Meeting, Aug. 25, 1994, published in the Issue of Chemical and Engineering News, 10 1994).
Biocode, Limited has used fluorescent labeled antibodies to determine ingredients in products.
U.S. Pat. No. 5,429,952 discloses adding light-emissive chemicals to a product for analysis, as exogenous product tags which do not ordinarily form part of the product.
The use of standard analytical methods to monitor every lot or batch for a product or competitor product for authenticity or compliance with laboratory equipment can often be costly.
I have discovered an automated method of developing a database to store information for xe2x80x9cfingerprintxe2x80x9d-type analysis of products (even as to product lot numbers and batch). The automated analysis is a method of evaluating and discriminating products, even within a narrow field or industry, competing and otherwise, e.g., to establish authenticity or point of origin of the product. The invention relates to a method for identifying analytes such as key ingredients and/or the relative amounts of analytes such as key ingredients in products. The method allows for authenticating and monitoring products for fraud and quality control using light emission. The invention also relates to light-emissive-compounds (e.g., including one or more light emissive compounds) which can be used to identify and quantitate the relative amounts of analytes in products.
In general, the methods pertain to obtaining an emission profile of a sample. An xe2x80x9cemission profilexe2x80x9d, as used herein, refers to data collected relating to emission, for example, emission intensity or time of emission. The data collected for the emission profile can be expressed in relative terms, e.g. relative emission intensity or time of relative emission, when data for the sample and a standard and/or a control are compared.
In one aspect, the invention features a method for determining relatedness of a sample to a standard known to be authentic or known to have at least one selected characteristic of authentic material. The method includes: a) providing a mixture of sample and least one light-emissive compound (xe2x80x9cLECxe2x80x9d); (b) irradiating the sample mixture with an irradiating wavelength of light; (c) monitoring at least one emitted wavelength of light (generated in response to the irradiating) to establish a sample emission profile; and (d) providing a standard fingerprint characteristic of a standard mixture; and (e) comparing the sample emission profile with the standard fingerprint to determine whether the sample is authentic. The standard mixture includes the standard and the light-emissive compound. The standard fingerprint is generated by irradiating several of the standard mixture with the irradiating wavelength and monitoring the emitted wavelength in response thereto.
In preferred embodiments, two and preferably three or more light-emissive compounds are employed, and a fingerprint profile of several light-emissive compounds is compared to the corresponding emission intensities for the sample. Most preferably, the light-emissive compounds emit light at nonoverlapping wave lengths, whereby multiple compounds can be added to the sample and/or standard at the same time.
In preferred embodiments, the method further includes: providing a background control mixture which includes the light-emissive compound without the sample or the standard; irradiating the background control mixture with the irradiating wavelength and monitoring the emitted wavelength in response thereto, to establish background emission; and determining the emission profile of the sample based on at least one difference between the emission of the control mixture and the emission of the sample mixture. It is preferred that the standard be a composition having a predetermined relative amount of a component characteristic of authentic material. The sample fingerprint is generated based on a first change in emission, determined by comparing the background emission and the emission from the sample mixture. The standard fingerprint is generated based on a second change in emissions, determined by comparing the background emission and the standard emission for each measurement. The comparing step includes comparing the first change in emission to the background adjusted fingerprint, e.g., to quantify relative amounts of sample component.
In another aspect of the invention, a method is provided for determining whether a product is authentic. A liquid sample of a test product is obtained and a light emissive compound then is added to the liquid sample to form a test sample. The light emissive compound interacts with an analyte of the product. The test sample is irradiated, and the intensity of light emitted from the test sample at a wavelength is determined. The intensity of light emitted from the test sample at this wavelength then is compared to the intensity of light emitted at the wavelength as a result of irradiating a mixture of the light emitting compound and an authentic liquid standard of the product, wherein similarity of light emission intensity is determinative of authenticity of the sample and this similarity of light emission intensity is determinative of nonauthenticity of the sample. In one important embodiment, the intensity of light emitted from the test sample is compared to the intensity of light emitted from a plurality of the mixture, and wherein authenticity requires the intensity of light emitted from the test sample to be within a pre-selected confidence limit defining a range of intensity calculated from the intensity of light emitted from the plurality of said mixture. The plurality of said mixture is at least four standards containing a mixture of the light emitting compound and an authentic liquid standard of the product, and preferably is four such mixtures.
In certain of the foregoing embodiments, the chemical composition of the product is unknown. In other of the embodiments, the chemical structure of the analyte to which the light emitting compound binds is unknown. In still other embodiments, the analyte is other than an exogenous product tag. In one particularly important embodiment, the product is a liquid consumable product.
As mentioned above, a plurality of light emissive compounds can be used. In such embodiments, it is preferred that each light emitting compound binds to a different analyte of the product. Most preferably, the light emissive compounds is a fluorescent dye.
In other preferred embodiments, the light-emissive compound is added to the sample by an automated pipette. It is preferred that the sample mixture be dispensed by an automated pipette in a multiwell plate.
In other preferred embodiments, the standard, the sample, or both, inherently include a fluorescent, phosphorescent, or luminescent compound. In some products the compound is caffeine.
In other preferred embodiments, the light-emissive compound is fluorescent, phosphorescent, or luminescent, and emission varies in response to quantity or quality of product analytes. Preferably, the light-emissive compound interacts with components of the sample, the standard, or both, to yield at least one fluorescent, phosphorescent, or luminescent component.
In other preferred embodiments, the standard is a composition having a predetermined relative amount of an analyte characteristic of authentic material, and the comparing step includes quantifying the relative amounts of the analyte in the sample.
In preferred embodiments, the method includes performing steps (b)-(c) described above, at least two times and preferably three times. Steps (b)-(c) may be performed using the same or different light-emissive compounds, and the same or different irradiating and emission wavelengths are monitored in each performed step.
In one important embodiment, the standard is a caffeine-containing beverage, and the light-emissive compound is: a) 5-(2-carbohydrazinomethylthioacetyl)aminofluorescein; b) 5-(4,6-dichlorotriazinyl)aminofluorescein; c)Fluo-3 pentaammonium salt (Minta et al., J. Biol. Chem. 264:8171, 1989 and U.S. Pat. No. 5,049,673); d) 4-aminofluorescein; e) 5-aminofluorescein; f) sulfite blue coumarin; g) courmarin diacid cryptand (CD222) (Costlei et al., J. of Chem. Society Perkins translation 2, p. 1615); or h) Eosin Y.
In another important embodiment, the standard is an infant formula, and the light-emissive compound is selected from the group consisting of 5-(2-carbohydrazinomethylthioacetyl) aminofluorescein, 5-(4,6-dichlorotriazinyl)aminofluorescein, Fluuo-3 pentaammonium salt, or Courmarin benzothiazole, tetrapotassium salt (BTC5N) (Cell Calcium, p. 190, 1994). In other preferred embodiments, the standard contains corn syrup, and the light-emissive compound is selected from the group consisting of 5-(2-carbohydrazinomethylthioacetyl) aminofluorescein, 5-(4,6-dichlorotriazinyl)aminofluorescein, Fluo-3 pentaammonium salt, 4-aminofluorescein, 5-aminofluorescein, sulfite blue coumarin, courmarin diacid cryptand (CD222), or Eosin Y. In other preferred embodiments, the standard is an ethanol-containing beverage and the light-emissive compound is selected from the group consisting of 5-(2-carbohydrazinomethylthioacetyl)aminofluorescein, 5-(4,6-dichlorotriazinyl)aminofluorescein, Fluo-3 pentaammonium salt, proflavine hemisulfate, tetra(tetramethylammonium) salt, acridine orange hydrochloride hydrate, BTC-5N, acriflavine, 4-aminofluorescein, or 5-aminofluorescein. Compound 11 is sulfite blue coumarin compound 12 is courmarin diacid cryptand (CD222). Compound 13 is Eosin Y. In other preferred embodiments, the standard is an aqueous mixture, and the light-emissive compound is a compound that interacts or reacts with heavy metals, the light-emissive compound being selected from the group consisting of Fluo-3 pentaammonium salt, or BTC-5N.
In another aspect, the invention features a method for determining relatedness of a first sample to a second sample, neither of which is a known standard. The method includes: (a) providing a first sample mixture including the first sample and at least one light-emissive compound; (b) irradiating a plurality of the first sample mixture with an irradiating wavelength of light; (c) monitoring at least one emitted wavelength of light generated in response to the irradiating, to establish a first sample fingerprint characteristic of the first sample mixture; (d) providing a second sample fingerprint characteristic of a second sample mixture, the second sample mixture including the second sample and the light-emissive compound; the second sample fingerprint being generated by irradiating a plurality of the second sample mixture with the irradiating wavelength and monitoring the emitted wavelength in response thereto; and (e) comparing the first sample fingerprint with the second sample fingerprint to determine relatedness of the two samples.
In preferred embodiments, the first sample is identified as a specific product or as part of a homogeneous lot of a product by comparing the fingerprint profile or emission profile of the first sample to a library of fingerprints of samples whose product composition or lot number are known.
In other preferred embodiments, the method further includes providing one or more additional fingerprints to generate a fingerprint profile for each of at least two additional light emissive compounds and comparing the first sample mixture fingerprint profile to the second sample or standard fingerprint profile.
In preferred embodiments, the method is used to determine product authenticity, product tampering or product manufacturing compliance. In other preferred embodiments, the sample is a perfume, fragrance, flavor, food, or beverage product.
In another aspect of the invention, a method is provided for selecting a dye for determining authenticity of a product. A candidate dye is added to a plurality of candidate dilutions of a liquid sample of an authentic standard of the product, the candidate dye being light emissive at a particular wavelength when irradiated if it interacts with an analyte in the liquid sample. A test dilution then is selected at which the candidate dye emits light at a selected intensity when said candidate dye is added to said liquid sample at the test dilution. A range of intensity of light emission at discrete wavelengths is determined for a plurality of mixtures of said candidate dye and said liquid sample at said test dilution. An experimental intensity of light emission at the discrete wavelengths is then determined for a mixture of said candidate dye and a liquid sample of nonauthentic product at said test dilution. Finally, the experimental intensity at the discrete wavelengths is compared to the range of intensity of light emission at the discrete wavelengths, said dye being selected as useful for determining authenticity of said product if said experimental intensity falls outside of said range of light emission at the discrete wavelengths. In one embodiment, the candidate dye is a plurality of candidate dyes, each of the dyes emitting light at different wavelengths, and wherein the analyte is a plurality of analytes, each dye binding to a different of said plurality of the plurality of analytes. In an important embodiment, the chemical composition of the product is unknown and/or the chemical structure of the analyte is unknown. In other important embodiments, the product is a liquid consumable product.
In another aspect of the invention, a computer implemented method for determining authenticity of a liquid product is provided. The method involves receiving light emission data produced by adding a component to a test sample of the liquid product and measuring light emission therefrom. It also involves receiving light emission data produced by measuring light emission from a sample of a mixture of an authentic liquid product and the component. There then is a comparison of the intensity of light emission from the test sample to intensity of light emission from samples of the plurality of the mixtures, wherein authenticity requires the intensity of light emission from the test sample to be within a preselected confidence limit defining a range of intensity at discrete wavelengths calculated from the intensity of light emission at the discrete wavelengths from the plurality of the mixtures.
In one important embodiment, a computer database is used for storing and making available information about light emission of an authentic product. The database includes a computer-readable medium having a computer-readable logic stored thereon, wherein the computer-readable logic comprises a plurality of records for the authentic product indicating measurements of intensity of light emitted by samples of a plurality of mixtures of the authentic product with a component. The database also includes an indication of the component, wherein the records are accessible using an indication of the component and/or the authentic product wherein the step of receiving light emission data for the authentic product includes the step of accessing the computer-readable medium using an indication of the component and/or the product to retrieve the records.
In another aspect of the invention, a computer database for storing and making available information about light emission of an authentic product is provided. The database included a computer-readable medium having computer-readable logics stored thereon, wherein the computer-readable logic comprises a plurality of records for the authentic product indicating measurements of intensity of light emitted by samples of a plurality of mixtures of the authentic product with a component, and an indication of the component. Also included are means for accessing the computer-readable medium using an indication of the component and/or the authentic product to retrieve the records.
It is a feature also of the present invention that, when adding a light-emitting compound to a sample in accordance with the methods described herein, the sample can be separate from the standard. This differs from the situation where product tags are used, in that product tags are added to an authentic product to form a tagged mixture wherein the addition of the tag to the sample is not separate from the addition of the tag to the standard.
Light-emissive compounds are involved in light emission in response to irradiation with light of a different wavelength. Light emission of interest can be a result of phosphorescence, chemiluminescence, or, more preferably, fluorescence or polarized fluorescence. Specifically, the term xe2x80x9clight emissive compounds,xe2x80x9d as used herein, means compounds that have one or more of the following properties: 1) they are a fluorescent, phosphorescent, or luminescent; 2) interact with components of the sample or the standard or both to yield at least one fluorescent, phosphorescent, or luminescent compound; or 3) interact with at least one fluorescent, phosphorescent, or luminescent compound in the sample, the standard, or both to alter emission at the emission wavelength. The emission wavelength can be any detectable wavelength including visible, infrared (including near infrared), and ultraviolet. Light, as used herein, likewise can be of any wavelength.
Light-emissive compounds also include compounds that cause, or interact with components of the standard or sample to cause, or alter, Raman Scatter at a scatter or emission wavelength. The Raman effect occurs when light from a strong source (typically a laser) interacts with a material. Most of the light is absorbed or scattered without wavelength change but some of the light is scattered into other wavelengths (the Raman scatter).
xe2x80x9cFingerprintxe2x80x9d refers to the data set of light emission intensity from a light-emissive compound in combination with a liquid sample of a product measured; at least three times, three such combinations measured at least once, or both. Accordingly, each product can have a particular fingerprint. A xe2x80x9cfingerprint profilexe2x80x9d is an assembly of fingerprints of a liquid sample of a product in combination with a series (or profile) of different light-emissive compounds.
As noted above, the emission profile can include, but is not limited to, emission intensity and time of relative emission. The same information that can be derived about the amount and/or concentration of analytes by emission intensity measurement also can be derived from measurement of the time of emission, e.g. the time of relative emission of fluorescent compounds in a sample. Analysis of the emission intensity or time of relative emission can be done as described herein and by other methods known to one of ordinary skill in the art. Other emission properties measurable by one of ordinary skill in the art (e.g. emission half-life, emission decay characteristics) are also embraced in the term emission profile.
The term xe2x80x9canalytexe2x80x9d, as used herein, means a key ingredient or trace compound of the product. A native analyte is one which is ordinarily found in the unadulterated product, not added as an exogenous product tag. The invention relies upon interaction of light emissive compounds with such analytes, whereby alterations in a product can be detected, including (1) dilution of an analyte, (2) substitution of an ingredient for an analyte, (3) addition of a compound which alters interaction of the light emissive compound with an analyte and (4) addition of a compound which quenches light emission resulting from interaction of a light emissive compound with an analyte. Most frequently the alteration detected is in the amount of analyte bound to the light emissive compound, which is reflected by the intensity of light emitted when a sample is irradiated.
By xe2x80x9cinteracts withxe2x80x9d, as used herein, it is meant reacting, intercalating, binding or any other interaction which causes the dye to alter its light emission properties when irradiated.
The term xe2x80x9ckey ingredient,xe2x80x9d as used herein, means a component included in a composition of a product that is important in identifying the particular product.
The term xe2x80x9ctrace compound,xe2x80x9d as used herein, means a compound that is present in low concentrations (e.g., at ppm or ppb levels) in a product. The trace compound can be related, for example, to a particular key ingredient. The trace compound can be introduced at the source of the key ingredient or during the manufacture of the product.
The invention can include one or more of the following advantages. The method can be used in the distilled spirits industry, where trace compounds and key ingredients can be measured using specific light-emissive compounds. Further, light-emissive compounds that indicate the source of ethanol can be used to determine the authenticity of a product. For example, spirits derived from yellow dent corn contain different trace compounds than spirits derived from cane sugar.
Moreover, although colas, and other soft drinks, contain similar levels of key ingredients, the levels key ingredients can be used to determine whether a particular manufacturer is diluting the concentrate to the appropriate level. For example, caffeine can be a targeted ingredient for light-emissive compounds in the analysis of soft drinks. Additional targets in soft drinks can include, but are not limited to, the high fructose corn syrup and the pH.
Furthermore, perfumes, fragrances, flavors, foods, and all types of beverages can be fingerprinted, using the methods of the invention, without adding any reagents to the product the user is going to consume. An advantage of invention is that exogenous product tags need not be added. Instead, native analytes of the product can be assayed. This is particularly important in determining authenticity of food products, where it is undesirable to add tags which could affect taste, odor, consistency and the like and might even be harmful to health when ingested. This is of great importance to many companies which are reluctant to adulterate their products.
The invention also is useful in identifying pharmaceutical active ingredients and/or excipients. The invention, therefore, can be used to authenticate pharmaceutical or other chemical products. In the instance where a fingerprint is obtained for a pharmaceutical formulation, an identical fingerprint may permit an inference that the pharmaceutical formulation was prepared by a particular process, which itself may be a patented process. In addition, there may be unique ingredients used in a patented process, the presence and concentration of which can be used to determine the authenticity of a material manufactured by that process (when the material contains trace levels of the unique ingredients) or as evidence of infringement of the patented process. Thus, single or multiple dyes can be selected or developed to identify compounds or excipients that would be present (or present at particular concentrations) only as a result of performing a patented process.
The invention allows accurate light-emissive profiles of products to be determined and monitored without altering the product.
Another advantage of the invention is that it is unnecessary to know or determine the composition of the product in order to select light emissive compounds and to develop assays for determining accurately authenticity. Thus, it is unnecessary to know or determine the formula for Coca-Cola(copyright) or Pepsi(copyright) in order to test the authenticity of products sold under those trademarks. This is to be contrasted with many infrared methods (e.g., near IR, mid IR and Fourier Transform IR), that often result in gathering sufficient information to determine the composition of a product being tested. This advantage of the present invention is of great importance to companies reluctant to identify the secret ingredients of their products.
A further advantage of the invention is that the use of light-emitting compounds results in a sensitivity level that far exceeds the sensitivity levels achievable by the use of Fourier Transform IR methods.
Other features and advantages of the invention will be apparent from the following detailed description thereof, and from the claims.